The long-term objective of this proposal is to understand the cellular mechanisms responsible for abnormal transepithelial NaCl and fluid secretion by cysts in human autosomal dominant polycystic kidney (ADPKD) kidneys. The ultimate goal is to identify an approach to slow or arrest the progression of ADPKD. To this end studies will be conducted to test the primary hypothesis that NaCl and fluid secretion by human ADPKD cysts is driven by electrogenic Cl secretion and that secretion is regulated by arginine vasopressin (AVP), adenosine and prostaglandins (PG) via an increase in cAMP. It is proposed that electrogenic Cl secretion involves uptake of Cl across the basolateral membrane by Na/K/2Cl cotransport and Cl/HCO exchange and diffusion out of the cells across the apical membrane by CFTR Cl channels. The Na-K-ATPase, by maintaining a low intracellular Na concentration, provides the driving force for Cl uptake across the basolateral membrane via the Na/K/2Cl cotransporter. Studies will also be conducted to test the hypothesis that intracellular trafficking of CFTR Cl channels in ADPKD cells is regulated by cAMP and is dependent on microtubules and the actin-based cytoskeleton. We propose that the observed changes in the cyoskeleton in ADPKD cells may alter the trafficking and insertion of CFTR Cl channels into the apical membrane. There are three specific aims. Specific Aim #1: To elucidate the cellular mechanisms of electrogenic Cl secretion across ADPKD epithelia. Transport mechanisms will be identified and characterized in human ADPKD cells using a number of experimental approaches including measurements of short circuit current, confocal-immunofluorescent microscopy and the patch clamp technique. Specific Aim #2: To elucidate the signal transduction pathways regulating Cl secretion across ADPKD epithelia. The regulation of Cl secretion by AVP, adenosine and PG and the signal transduction pathways mediating Cl secretion by these hormones will be evaluated using the patch clamp technique and measurements of short circuit current. Specific Aim #3: To elucidate the intracellular trafficking of CFTR Cl channels in ADPKD epithelia. Intracellular trafficking of CFTR and the regulation of trafficking by microtubules and the actin-based cytoskeleton will be evaluated by time-lapse, video-confocal microscopy of a CFTR-green fluorescent protein (GFP) fusion protein transfected into human ADPKD cells. An important aspect of this application is that the proposed studies will be performed on human ADPKD cells. Thus, the information derived from-these studies will provide a comprehensive and integrative understanding of the cellular mechanisms mediating fluid secretion into the lumen of cysts in human ADPKD kidneys and may ultimately lead to the identification of an approach to slow or arrest the progression of ADPKD.